Diabetes mellitus in early pregnancy causes birth defects, even when perinatal care is available. Birth defects produce life-long effects on the children and extraordinary stress on healthcare resources. This is a serious public health crisis in the United States where the number of diabetic patients, including women in child-bearing age, is increasing rapidly. The common anomalies resulted from diabetic pregnancy are in the central nervous system, primarily neural tube defects (NTDs). NTDs are caused by excessive programmed cell death (apoptosis) in the neural epithelium, which is executed by initiator caspase-8-triggered apoptotic signaling. Caspase-8 is activated by a protein complex, known as death inducing signaling complex (DISC) involving TRAF1 and TRADD. However, how hyperglycemia activates DISC/caspase-8 remain to be delineated. Maternal hyperglycemia disturbs glucose metabolism (glycolysis) in the embryo, augmenting the hexosamine biosynthetic pathway (HBP) to produce high levels of uridine diphosphate N-acetylglucosamine (UDP-GlcNAc). Catalyzed by O-linked-GlcNAc transferase (OGT), a large number of proteins are O- GlcNAcylated. This posttranslational modification alters the function of chaperone proteins, which facilitate folding and processing of newly synthesized polypeptides in the endoplasmic reticulum (ER) and transporting out of its lumen. The consequent prolonged retention of proteins in the ER produces stress conditions, referred to as ER stress. ER stress activates so-called unfolded protein response (UPR) to resolve protein folding crisis and alter cellular activities. UPR increases the expression of C/EBP homologous protein (CHOP), which, in turn, up-regulates pro-apoptotic factors to trigger apoptosis. We hypothesize that maternal hyperglycemia promotes O-GlcNAcylation of chaperone proteins, resulting in impaired protein folding/processing, which, in turn, activates UPR/CHOP leading to caspase-8-initiated apoptosis and eventually NTDs. To test our hypothesis, we will investigate each component of the GlcNAcylation-protein folding-UPR-DISC- capsase8 cascade to delineate underlying mechanisms and gain insight into potential interventional approaches. In Specific Aim 1, we will systematically examine protein O-GlcNAcylation to identify important protein families and associated biological processes in diabetic embryopathy. We will investigate the mechanism underlying chaperone proteins in regulating protein folding. We will target protein O-GlcNAcylation and folding/processing using OGT inhibitors and chemical chaperones, respectively, to identify their role in diabetic embryopathy and test potential interventional approaches. In Specific Aim 2, we investigate the role of a key UPR factor CHOP in embryonic malformation using a chop gene knockout mouse model, and delineate underlying cellular and molecular mechanisms. In Specific Aim 3, we will investigate whether TRAF1 triggers DISC formation by binding to TRADD and other DISC factors. We will, then, investigate whether TRAF1 causes NTDs in diabetic embryopathy using a traf1 knockout model.